Abstract: Embryonic stem (ES) cell research holds great promise for the future of medicine. In particular, new insights into human development, the etiology of complex diseases, as well as novel therapies will emerge. Fulfillment of these promises depends on comprehensive understanding of the regulatory processes that control cell-fate decisions in ES cells. Many years of work with mouse ES cells have defined a number of regulatory molecules and signaling pathways that are required for self-renewal, and lineage-specific differentiation. However, much remains to be learned. Comparisons of mouse and human ES cells have revealed similarities, as well as differences. We have developed novel approaches for global functional genomic analyses of cell fate regulation in mouse ES cells. Here, we will apply these approaches to define a comprehensive functional "parts list" of molecules responsible for control of cell-fate decisions. Our strategies are based on short hairpin RNA-mediated loss-of-function analyses of candidate regulatory gene- products. Perturbation of individual molecules allows multi-level analyses of cell fate changes, as they occur in time. We will apply a systems biology strategy an analyze cell-fate control simultaneously at the epigenetic, transcriptional, and proteomic levels. Coupled with genome-wide technologies, such perturbations also allow provisional assembly of the "parts" into transcriptional, as well as other types of regulatory networks. Our major focus will be on the control of ES cell self-renewal. Subsequently, we will initiate efforts to dissect the mechanisms that control commitment to specific cell lineages. Our results will provide a firm foundation for understanding ES cell fate regulation in a deep, systems level manner. They will also provide a framework and the tools for comprehensive analyses of cell-fate regulation in the human ES cell system. Ultimately, our studies will also provide reliable and robust avenues to direct ES cell differentiation to specific cell types and populations.